Oxidative stress plays a major role in the pathogenesis of many human diseases, and in particular, neurodegenerative diseases. Treatment with antioxidants, which may reduce particular free radical species, therefore, might theoretically prevent tissue damage and improve both survival and neurological outcome. Free radicals in physiological environments can often be classified as either a reactive oxygen species (ROS) or a reactive nitrogen species (RNS). Free radicals are highly reactive chemical species and readily react with proteins, lipids and nucleic acids at a subcellular level and thereby contribute to the progression of various diseases and events producing oxidative stress, such as ischemic stroke.
The origin of the use of nanoceria in nano-medicine can be traced to the seminal work of Bailey and Rzigalinski, wherein the application of ultrafine cerium oxide particles to brain cells in culture was observed to greatly enhanced cell survivability, as described by Rzigalinski in Nanoparticles and Cell Longevity, Technology in Cancer Research & Treatment 4(6), 651-659 (2005). More particularly, rat brain cell cultures in vitro were shown to survive approximately 3-4 times longer when treated with 2-10 nanometer (nm) sized cerium oxide nanoparticles synthesized by a reverse micelle micro emulsion technique, as disclosed by Rzigalinski et al. in U.S. Pat. No. 7,534,453, filed Sep. 4, 2003. Cultured brain cells exposed to a lethal dose of free radicals generated by hydrogen peroxide or ultraviolet light exposures were afforded considerable protection by the cerium oxide nanoparticles. In addition, the cerium oxide nanoparticles were reported to be relatively inert in the murine body, with low toxicity (e.g. tail vein injections produced no toxic effects). While no in vivo medical benefits were reported, benefits were postulated for treatments with these ceria nanoparticles, including reduced inflammation associated with wounds, implants, arthritis, joint disease, vascular disease, tissue aging, stroke and traumatic brain injury.
However, a host of problems with these particular nanoceria particles was subsequently disclosed by Rzigalinski et al. in WO 2007/002662. Nanoceria produced by this reverse micelle micro emulsion technique suffered from several problems: (1) particle size was not well-controlled within the reported 2-10 nanometer (nm) range, making variability between batches high; (2) tailing of surfactants, such as sodium bis(ethylhexyl)sulphosuccinate, also known as docusate sodium or (AOT), used in the process into the final product caused toxic responses; (3) inability to control the amount of surfactant tailing posed problems with agglomeration when these nanoparticles were placed in biological media, resulting in reduced efficacy and deliverability; and (4) instability of the valence state of cerium (+3/+4) over time. Thus, the cerium oxide nanoparticles produced by the reverse micelle micro emulsion technique were highly variable from batch to batch, and showed higher than desired toxicity to mammalian cells.
As an alternative, Rzigalinski et al. in WO 2007/002662 describe the biological efficacy of nanoceria synthesized by high temperature techniques, obtained from at least three commercial sources. These new sources of cerium oxide nanoparticles were reported to provide superior reproducibility of activity from batch to batch. It was further reported that, regardless of source, cerium oxide particles having a small size, narrow size distribution, and low agglomeration rate are most advantageous. In regard to size, this disclosure specifically teaches that in embodiments where particles are taken into the interior of cells, the preferable size range of particles that are taken into the cell are from about 11 nm to about 50 nm, such as about 20 nm. In embodiments where particles exert their effects on cells from outside the cells, the preferable size range of these extracellular particles is from about 11 nm to about 500 nm.
These inventors (Rzigalinski et al.) also report that for delivery, the nanoparticles were advantageously in a non-agglomerated form. To accomplish this, they reported that stock solutions of about 10% by weight could be sonicated in ultra-high purity water or in normal saline prepared with ultra-high purity water. However, as others have noted, we have observed that sonicated aqueous dispersions of nanoceria synthesized by high temperature techniques (e.g. obtained from commercial sources) are highly unstable, and settle rapidly (i.e. within minutes), causing substantial variability in administering aqueous dispersions of nanoceria derived from these sources.
Hardas et al., Toxicological Sciences 116(2), 562-576 (2010), report on the biodistribution and toxicology effects of aqueous dispersions of nanoceria prepared by the direct two-step hydrothermal preparation of Masui et al., J. Mater. Sci. Lett. 21, 489-491 (2002), in which sodium citrate is included as a biocompatible stabilizer. High resolution TEM revealed that this form of nanoceria possessed crystalline polyhedral particle morphology with sharp edges and a narrow size distribution of 4-6 nm. These citrate stabilized ceria nanoparticle dispersions were reported to be stable for more than 2 months at a physiological pH of 7.35. Thus, no sonication prior to administration was required.
Quite surprisingly, however, they report that compared with previously studied commercially sourced nanoceria (Aldrich Chemical Co. (Cat. #639648)), this form of citrate stabilized nanoceria was more toxic, was not seen in the brain, and produced little oxidative stress effect to the hippocampus and cerebellum.
DiFrancesco et al. in commonly assigned PCT/US2007/077545, METHOD OF PREPARING CERIUM DIOXIDE NANOPARTICLES, filed Sep. 4, 2007, describes the oxidation of cerous ion by hydrogen peroxide at low pH (<4.5) in the presence of biocompatible stabilizers, such as citric acid (CA), lactic acid, tartaric acid, gluconic acid, ethylenediaminetetraacetic acid (EDTA), and combinations thereof. Specifically, the stabilizer lactic acid and the combination of lactic acid and EDTA are shown to directly produce stable dispersions of nanoceria of average particle size in the range of 3-8 nm.
Reed et al. in commonly assigned US2013/0337083, NANOCERIA FOR THE TREATMENT OF OXIDATIVE STRESS, filed Mar. 15, 2013, describes a synergistic increase in hippocampal cell sparing for mice treated with nanoceria prepared with a combination of citric acid (CA) and EDTA, wherein the molar ratio of CA/EDTA ranges from 3.0 to 0.1. Amelioration of disease progression and improvement in motor behavior tests resulted in murine models of chronic-progressive multiple sclerosis, relapse-remitting multiple sclerosis, amyotrophic lateral sclerosis and ischemic reperfusion injury.
As described previously, various methods have been reported for preparing biocompatible dispersions of cerium-containing nanoparticles, and in particular those stabilized with citric acid or citrate ion. However, a need remains for further improvement in the free radical scavenging ability of citric acid stabilized cerium-containing nanoparticle dispersions, used, for example, to treat the effects of oxidative stress related diseases and events, such as ischemic stroke.